Design of hormone-like antibodies with agonistic and antagonistic functions

ABSTRACT

The present invention relates to an agonistic anti-human TrkA mAb 5C3 which recognizes the NGF docking site. Such antibodies may be used for the treatment, diagnosis or prevention of neurological diseases, neuromas and neoplastic tumors which express TrkA receptors. Also these antibodies may be used to develop and screen for pharmaceutical agents which are agonistic or antagonistic to NGF binding to the TrkA receptors.

This application is the 35 U.S.C. §371 National Phase of PCT/CA96/00815,filed Dec. 6, 1996, which claims priority of GB 9525180.7, filed Dec. 8,1995.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The invention relates to a method of designing agonistic and/orantagonistic antibodies for any hormone receptor more specificallyantibodies which are capable of blocking nerve growth factor (NGF)binding and antibodies which can mimic NGF binding to its receptor.

(b) Description of Prior Art

The TrkA receptor is a 140 kDa transmembrane glycoprotein with tyrosinekinase activity that functions as the Nerve Growth Factor (NGF)receptor. NGF also binds with low affinity to a p75 receptor whosesignaling function is unclear. Homo or heterodimers or oligomers of TrkAand p75 bind NGF with higher affinity (Jing, S. et al. (1992) Neuron, 9:1067-1079) suggesting that specific receptor conformations may playspecific functions.

NGF promotes the differentiation of certain neuronal cells, is mitogenicfor TrkA-transfected fibroblasts, and allows survival in serum-deprivedconditions for both cell types. Activation of the tyrosine kinaseactivity of TrkA via UGF binding leads to receptor trans- andauto-tyrosine phosphorylation (PY), and PY of second messengersincluding phosphatidylinositol-3 kinase (PI-3 kinase). PI-3 kinase isinvolved in protein trafficking and endocytosis of ligand-receptorcomplexes (reviewed by Kaplan, D. R. et al. (1994) J. Neurobiol., 25:1404-1417). Since microinjection of NGF into cells does not result inNGF biological signals, cell surface receptor ligation andinternalization of TrkA or NGF-TrkA complexes must mediate theseeffects.

TrkA, like most kinase growth factor receptors, signals through receptoroligomerization. Thus, mono-valent TrkA-binding agents are antagonisticor have no biological effects (LeSauteur, L. et al.(1995) J. Biol.Chem., 270:6564-6569), whereas bivalent receptor-binding agents such asNGF (a homodimer) or antibodies can be agonistic. The principle of usingpolyclonal antibodies to activate neural receptors has been demonstrated(Twyman, R. E. et al. (1995) Neuron, 14: 755-762). In contrast, only alimited number of anti-receptor monoclonal antibodies (mAb) mimic ligandfunctions (Taub, R. et al. (1992) Biochemistry, 31: 7431-7435), and noneexist against neurotrophin receptors.

It would be highly desirable to be provided with an agonistic orantagonistic anti-human TrkA mAb which recognizes the NGF docking site.Such antibodies may be used for the diagnosis, treatment or preventionof neurological diseases, neuromas and neoplastic tumors which expressTrkA receptors. Also these antibodies may be used to develop and screenfor pharmaceutical agents which are agonistic or antagonistic by bindingto the TrkA receptors.

SUMMARY OF THE INVENTION

One aim of the present invention is to report the development andcharacterization of an agonistic anti-human TrkA mAb 5C3 whichrecognizes at least one NGF docking site. This MAb 5C3 was used tocharacterize the pattern of TrkA protein expression in normal humanbrain, and the NGF binding features of the receptor. MAb 5C3 behaveslike NGF in bioassays, and monomeric 5C3 F_(abs) retained binding andfunctional agonistic activity. MAb 5C3 will be useful to identify theNGF docking site on TrkA and possibly as a pharmacological lead in thedevelopment of small mimetics.

In accordance with the present invention there is provided an antibodyor functional fragment thereof which binds to at least the TrkA receptorunder physiological conditions, and wherein the binding to the receptorat least partially mimics or inhibits nerve growth factor biologicalactivity.

In accordance with the present invention there is also provided a methodof screening pharmacological agents which are capable to mimic orinhibit nerve growth factor biological activity, which comprises usingthe antibody of the present invention to screen for pharmacologicalagents capable of binding to the complementary determining region of theantibody, wherein the screened pharmacological agents can mimic orinhibit nerve growth factor biological activity.

In accordance with the present invention there is also provided the useof pharmacological agents obtained by the process of the presentinvention, for the in vivo inhibition of nerve growth factor binding toTrkA receptor or the internalization or downmodulation of the receptor.

In accordance with the present invention there is also provided the useof the antibody of the present invention, for the in vivo inhibition ofnerve growth factor binding to TrkA receptor or the internalization ordownmodulation of the receptor, such as for inhibiting tumor growth insitu, for the treatment or prevention of neurological diseases, neuromasand neoplastic tumors which express TrkA receptors, for mappinghormone-receptor interactive sites and receptor domain-functioncorrelation such as mapping TrkA docking sites, for screeningpharmacological agents which bind to the hormone-receptor interactivesites.

In accordance with the present invention there is also provided anantibody having CDR-like domains of hormones, wherein the antibody orfunctional fragment thereof binds to at least TrkA receptor underphysiological conditions, and wherein the binding to the receptor atleast partially mimics or inhibits nerve growth factor biologicalactivity.

In accordance with the present invention there is also provided a methodfor the treatment of neurological diseases, neuromas and neoplastictumors which express TrkA receptors in a patient, which comprisesadministering an effective amount of an antibody of the presentinvention or a functional fragment thereof to a patient.

In accordance with the present invention there is also provided apharmaceutical composition for the treatment of neurological diseases,neuromas and neoplastic tumors which express TrkA receptors, whichcomprises an effective amount of an antibody of the present invention ora functional fragment thereof in association with a pharmaceuticallyacceptable carrier.

In accordance with the present invention there is also provided a methodfor immunization of a mammal against an antibody of the presentinvention or a functional fragment thereof, which comprisesadministering by systemic injection an immunizing amount of at least oneof the antibody or the fragment thereof in an immunogenic form inassociation with a pharmaceutically acceptable carrier.

In accordance with the present invention there is also provided a methodfor the prognosis or diagnosis of human tumors which comprises:

a) biopsy and immunocytochemistry of tumors using the antibody of thepresent invention and fragments thereof; or

b) radiolabeling of the antibody of the present invention and fragmentsthereof and nuclear imaging analysis.

In accordance with the present invention there is also provided a methodfor the treatment of human tumor of a patient which comprises the stepsof:

a) coupling cytotoxic agents to the antibody of the present inventionand fragments thereof;

b) administering the coupled antibody of step a) to the patient.

In accordance with the present invention there is also provided apharmaceutical composition for the targeting of pharmaceutical agents totissues of the central and/or peripheral nervous system, which comprisesan effective amount of an antibody of the present invention or afunctional fragment thereof coupled to a pharmaceutical agent inassociation with a pharmaceutically acceptable carrier. Thepharmaceutical agent may be selected from the group consisting ofradioligands, nucleic acid molecules, toxins, growth factors andgangliosides.

In accordance with the present invention there is also provided a TrkAdocking site which binds to the antibody of the present invention.

In accordance with the present invention there is also provided the useof the docking site of the present invention for screeningpharmacological agents which are capable to mimic or inhibit nervegrowth factor biological activity and said antibody biological antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (FIG. 1A, FIG. 1B) illustrates surface immunofluorescence studieswith mAb 5C3;

FIG. 2 (FIG. 2A, FIG. 2B) illustrates the direct detection of p140 TrkAby western blotting;

FIG. 3 (FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H) illustrates TrkAimmunoreactivity in normal human brain;

FIG. 4 illustrates the induction of TrkA-tyrosine phosphorylation by5C3;

FIG. 5 illustrates 5C3 binding studies and Scatchard plot analysis;

FIG. 6 illustrates the protection from apoptotic death by 5C3 and 5C3Fabs;

FIG. 7 illustrates nuclear imaging of tumors in vivo with 5C3;

FIG. 8 illustrates the survival of TrkA-expressing cells in serum-freemedia by 5C3 and derivatives;

FIG. 9 illustrates the differentiation/neurito-genesis of humanTrkA-expressing cells in serum media;

FIG. 10 illustrates Mab5C3 prevents TrkA-expressing tumor growth invivo; and

FIG. 11 illustrates the topography of the CDRs of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Monoclonal antibody (mAb) 5C3 directed against human p140 TrkA is astructural and functional mimic of NGF and an artificial receptoragonist. MAb 5C3 binds in the Nerve Growth Factor (NGF) docking site,and like NGF it promotes TrkA internalization; TrkA andphosphatidylinositol-3 kinase tyrosine phosphorylation; and increasedtransformation of TrkA-expressing fibroblasts. More importantly, mAb 5C3protects human TrkA-expressing cells from apoptotic death in serum-freemedia. Interestingly, agonistic activity is observed with monomericF_(ab) 5C3 fragments. The affinity of mAb 5C3 is ˜2 nM and was used tostudy features of ligand binding by TrkA and the distribution of TrkAprotein in normal human brain.

Antibodies

Female Balb/c mice were immunized with human TrkA, and splenocytes fusedto SP2/0 myelomas. Hybridomas were screened by differential bindingbetween untransfected and TrkA-transfected cells using a FluorescentActivated Cell Scanner™ (FACScan) (Becton Dickinson, Calif.). MAb 5C3was identified and subcloned 3 times. Rat anti-mouse IgG (amIgG) (Sigma,Saint Louis, Mo.), anti-phosphotyrosine mAb 4G10 (UBI, Lake Placid,N.Y.), and anti-PI-3 kinase polyclonal serum (UBI) were purchased, mouseanti-rat p75 mAb MC192 ascites were a gift from P. Barker, and anti-p65mAb 87.92.6 was grown in the laboratory.

Monomeric mAb 5C3 F_(abs)

MAb 5C3 was purified (1 mg/ml) with Protein G-Sepharose™ (Sigma) anddigested with papain (10 μg/ml) (GIBCO, Toronto, Ontario). F_(abs) wererepurified on KappaLock-Sepharose™ (UBI) and Protein G-Sepharose™ anddialyzed against phosphate buffered saline (PBS). All products werecharacterized by SDS-PAGE under non-reducing or reducing conditions (100mM 2-mercaptoethanol) to >98% purity. Control F_(abs) from anti-rat p75mAb MC192 were similarly prepared.

Cell Lines

Mouse SP2/0 myelomas, mouse R1.1 and EL4 thymomas, mouse NIH-3T3fibroblasts, mouse 2B4 T cell hybridomas, NGF responsive-rat PC12pheochromocytoma cells, human Jurkat T lymphomas, and human HeLafibroblasts were used. NIH-3T3 cells transfected either with humanp140trkA cDNA (E25 cells), with p75 cDNA (Z91 cells), or p75 andp140tzkA cDNAs (R7 cells) (Jing, S. et al. (1992) Neuron, 9: 1067-1079).The trk negative rat B104 neuronal cell line (expressing endogenous ratp75), and B104 transfected with human trkA CDNA (4-3.6 cells, expressinghuman TrkA and rat p75) were kindly provided by Dr. E. Bogenmann(Bogenmann, E. et al. (1995) Oncogene, 10: 1915-1925). PC12 cellstransfected with human trkA CDNA were obtained from Dr. Kaplan. Allcells were cultured in RPMI media supplemented with 5-10% Fetal BovineSerum (FBS) and antibiotics (GIBCO). Transfectants were added theappropriate drug selection.

FACScan

5×10⁵ cells in 0.1 ml of binding buffer (Hanks' Balanced Salt Solution(HBSS), 0.1% BSA, 0.1% NaN₃) were incubated with the indicatedconcentration of mabs or F_(abs) for 30 min at 4° C., washed in bindingbuffer to remove excess primary antibody, and immunostained withfluorescinated (FITC) goat anti-mouse IgG (FITC-GαmIgG), or anti-mouseFab (FITC-GαmF_(ab)) (Sigma) secondary antibody for 30 min at 4° C.Cells were acquired and analyzed on a FACScan (Becton Dickinson, Calif.)using the LYSIS II™ program. As negative controls (backgroundfluorescence) either mouse IgG (Sigma), mAb 192 or 192 F_(abs) were usedas primary, followed by appropriate secondary.

Characterization of mAb 5C3

FACScan analysis of non-permeabilized cells demonstrated that mAb 5C3recognizes the extracellular domain of human TrkA receptors (Table 1,FIG. 1A).

TABLE 1 Surface phenotyping with mAb 5C3 CELLS 5C3 BINDING E25 (hTrkA)+++++ R7 (hTrkA/p75) +++ Z91 (p75) − 4-3.6 (hTrkA/p75) +++ B104 (p75) −PC12 (rTrkA/p75) − transient NIH-3T3 transfections htrkA cDNA ++ htrkBcDNA − rtrkB cDNA − rtrkA cDNA −

The indicated cell lines expressing human TrkA (hTrkA), rat TrkA (rTrkA)and/or p75 were analyzed by surface immunofluorocytometry with mAb 5C3versus control mIgG. Transient transfections (48 hrs) were done byelectroporation of cDNAs. Relative intensities of staining are indicatedas +++++ (high staining) or − (no staining) (see FIG. 1). Saturatingdoses of mAb were used, and differences represent receptor number. Othercells tested include wild type NIH-3T3, Jurkat, R1.1, EL4, 2B4, and HeLacells which are all negative.

Human TrkA-transfectant lines 4-3.6, E25, and R7, bound mAb 5C3. Incontrast, rat PC12 (expressing rat TrkA and rat p75), rat B104 (parentalcells of 4-3.6, expressing rat p75), Z91 (NIH-3T3 transfected with p75),wild type NIH-3T3, or NIH-3T3 cells transiently transfected with humantrkB, rat trkA, or rat trkB cDNA did not bind mAb 5C3. Thus, mAb 5C3 isspecific for human TrkA, and co-expression of rat or human p75 does notinterfere with binding.

The concentration of mAb 5C3 required to saturate TrkA receptors wasdetermined in E25 cells by testing increasing amounts of antibody inFACScan assays (FIG. 1A). Similar analysis with mAb 5C3 F_(abs)demonstrated that specificity and saturability were similar to thatobtained with intact mAb but that 3-fold lower F_(ab) proteinconcentrations were required (FIG. 1B). Since the molecular weight of5C3 F_(ab) is 3-fold lower than 5C3 IgG (˜50 versus ˜150 kDa), equimolarconcentrations were required for saturation.

E25 cells expressing human TrkA were analyzed by indirectimmunofluorescence in a FACScan with various doses of mAb 5C3 or 5C3F_(abs) to assess ligand concentrations that achieve receptorsaturation.

(FIG. 1A) mAb 5C3 doses: 0.02 μg/ml (thick line, c); 0.2 μg/ml (thinline, b); 2 μg/ml (dotted line, a). For background fluorescence mIgG at2 μg/ml (crossed line, d) was used. (FIG. 1B) 5C3 F_(abs) doses: 0.007μg/ml (thick line, c); 0.07 μg/ml (thin line, b); 0.7 μg/ml (dottedline, a). For background fluorescence 192 F_(ab) at 0.7 μg/ml (crossedline, d) was used. Increased fluorescence intensity (X axis ofhistograms) reflect increased staining by mAb 5C3 or 5C3 F_(abs). Theareas under the curves represent the total number of cells acquired foreach sample (constant 5000 cells). Histogram heterogeneity is due toindividual cell receptor density.

Western blot analysis with mAb 5C3 revealed heterogeneous material ofM_(r) 140,000 (p140) for samples from E25 and 4-3.6 cells, but not forcontrol cells (FIG. 2A). In these cells a band of ˜110 kDa (p110) wasalso observed, previously thought to be intracellular TrkA precursors.The p140 band was also immunoblotted in samples dissected from normalhuman cortex or nucleus basalis of Meynert (FIG. 2B). The

110 band was not seen perhaps due to different post-translationalprocessing in neuronal tissues with respect to transfected cell lines.MAb 5C3 was effective in western blot analysis only when samples wereprepared under non-reducing or mildly reducing conditions, indicatingthat a disulfide bond-stabilized conformational epitope is recognized.

Whole cell detergent lysates (2×10⁶ cell equivalents/lane) were resolvedby SDS-PAGE under non-reducing conditions and analyzed by westernblotting with mAb 5C3. (FIG. 2A) Lane 1, Jurkat; lane 2, PC12; lane 3,NIH-3T3; lane 4, R1.1 ; lane 5, Z91; lane 6, E25. Thick arrow, p140TrkA; thin arrow, p110. (FIG. 2B) Dissected human brain tissues: nucleusbasalis (NB; lanes 1, 2, 6, 7), and cortex (FIG. 2C; lanes 3, 4, 8, 9)were compared to E25 cells (lane 5; 2×10⁵ cell equivalents). Lanes 1 and3, 300 μg/lane; lanes 2 and 4, 150 μg/lane; lanes 6 and 8, 75 μg/lane;lanes 7 and 9, 33 μg/lane. Thick arrow, p140 TrkA; thin arrow, p110.Note that p110 is not seen in the human brain tissues.

Biochemical Analysis

Cell Lysates

33×10⁶ cells/ml were detergent solubilized (lysis buffer 2% NP-40™, 150mM NaCl, 50 mM Tris-Glycine, 10 mM NaF, 50 μM Na₃VO₄, 30 mM NaPyrophosphate 10 mM benzamidine, 20 mM iodoacetamide, pH 7.8)supplemented with protease inhibitors (2 μg/ml soybean trypsininhibitor, 10 μg/ml aprotinin, 5 mM PMSF, and 10 μg/ml leupeptin) for 30min at 4° C., followed by a 15 minute centrifugation at 14,000 g.Cleared supernatants were analyzed by SDS-PAGE directly (whole celllysates) or after immunoprecipitation.

Gel Analysis

Cell lysates were prepared in Laemmli electrophoresis sample buffer andanalyzed by SDS-PAGE under reducing (100 mM 2-mercaptoethanol) ornon-reducing conditions. Prestained protein markers (GIBCO) were used asreference. Protein concentrations were quantitated by the biuret assay(Bio-Rad, Melville, N.Y.), and by parallel Coomassie blue staining ofSDS-PAGE gels. For western blotting samples were electrotransfered toPVDF (Xymotech Biosystems, Mt. Royal, QC), blocked overnight in TBST(0.05 M Tris base, 0.2 M NaCl, 0.5% Tween™-20, pH 7.6) containing 1% BSA(Sigma), and immunoblotted with the indicated primary mAbs. Secondaryantibodies were either horseradish peroxidase (HRP) conjugated goatanti-rabbit IgG (HRP-GαR), or goat anti-mouse IgG (HRP-GαM) (Sigma). Fordetection the enhanced chemiluminescence (ECL) reagents (Amersham,Oakville, Ont.) were used following the manufacturer's instructions.Densitometric analysis was performed with a Masterscan InterpretiveDensitometer CSPI™ and a Howtek Scanmaster™ (Scanalytics, Billerika,Mass.).

Binding, Competition, and Internalization Assays

MAb 5C3 was ¹²⁵I-labeled by the Iodogen (Pierce) method (Harlow, E.,Lane, D. (1988) A Laboratory Manual. Cold Spring Harbour LaboratoryPublishing. Chapter 9:332-333) to a specific activity of 1.8 mCi/mg.¹²⁵I[5C3] was repurified from free ¹²⁵I with Sephadex G25 columns (15×1cm) to >96% trichloroacetic acid precipitable incorporation. Bindingstudies were performed with serial dilutions of ¹²⁵I[5C3] on 0.5×10⁶ E25or 4-3.6 cells (and their respective controls NIH-3T3 and B104 cells)for 1 hour at 4° C. Cell-associated ¹²⁵I[5C3] and free ¹²⁵I[5C3] werecounted after washing unbound ligand. Parallel ¹²⁵I[NGF] (70 mCi/mg)(NEN-DuPont, Mississauga, Ont.) binding assays were performed ascontrol. Competition of ¹²⁵I[5C3] binding was done in binding assays inthe presence of unlabeled mAb 5C3 (100-fold molar excess), or unlabeledNGF (500-fold molar excess).

Competition of ¹²⁵I[NGF] binding was performed by first incubating cellswith excess unlabeled mAb 5C3, NGF, mAb 87.92.6 or vehicle bindingbuffer for 30 min at 4° C. ¹²⁵I[NGF] was then added to a finalsaturating concentration of ˜1 nM, the mixtures incubated for anadditional 45 min at 4° C., cells were washed and cell-associated125I[NGF] were determined.

Based on the competition assays above, it was desirable to identify themAb 5C3 receptor docking site which is presumed to be a functional orenergy-favorable site where the ligand-binding causes receptor-mediatedsignals.

This view is supported by similar findings in the EPO receptor system(Wrighton et al., 1996, Science, 273:458) wherein a “docking hot spot”was found; and by our finding that mAb 5C3 and NGF block each other'sbinding.

The docking site of mAb 5C3 was identified within the juxtamembrane/IgG2domain of human TrkA receptors.

The sequence is as follows:

Gln Val Asn Val Ser Phe Pro Ala Ser Val Gln Leu His Thr Ala Val (SEQ IDNO:9) 1               5                   10                  15 Glu MetHis His Trp Ser Ile Pro Phe Ser Val Asp Gly Gln Pro Ala            20                  25                  30 Pro Ser Leu ArgTrp Leu Phe Asn Gly Ser Val Leu Asn Glu Thr Ser        35                  40                  45 Phe Ile Phe Thr GluPhe Leu Glu Pro Ala Ala Asn Glu Thr Val Arg    50                  55                  60 His Gly Cys Leu Arg LeuAsn Gln Pro Thr His Val Asn Asn Gly Asn65                  70                  75                  80 Tyr ThrLeu Leu Ala Ala Asn Pro Phe Gly Gln Ala Ser Ala Ser Ile                85                  90                  95 Met Ala AlaPhe Met Asp Asn Pro Phe Glu Phe Asn Pro Glu Asp Pro            100                 105                 110 Ile Pro Asp ThrAsn Ser Thr Ser Gly Asp Pro Val Glu Lys Lys Asp        115                 120                 125 Glu Thr Pro     130

For receptor internalization studies cells were incubated withTrkA-binding agents (0.01 μg mAb 5C3; 2 nM NGF) or controls (mIgG; HBSS)for 20 min either at 37° C. (internalization permissive temperature) orat 40° C. (internalization non-premissive temperature). After washing,cells were processed for surface TrkA immunofluorescence with mAb 5C3primary and FITC-GαmIgG secondary as above, and analyzed by FACScan.

Binding Studies

Scatchard plot analysis of ¹²⁵I[5C3] binding assays demonstrated that inthe E25 cell surface there are ˜250,000 5C3 binding sites/cell with aK_(d) of 1.6 nM (FIG. 5), and in the 4-3.6 cell surface there are˜200,000 5C3 binding sites/cell with a K_(d) of 3.0 nM.

Serial dilutions ¹²⁵I[5C3] without competition (open triangles) wereused in binding studies with a constant number of E25 cells. Binding wascompeted with molar excess of unlabeled NGF (filled circles) or mAb 5C3(open squares). In 3 independent experiments the average K_(d) of mAb5C3 in E25 cells was 1.6 nM. Competition with NGF reduced the number of5C3 binding sites in E25 cells but the affinity of mAb 5C3 was notaffected.

No ¹²⁵[5C3] binding was observed for parental NIH-3T3 or B104 cells. NGFcompetition reduced the number of 5C3 binding sites in E25 cells by 25%.However, NGF caused no change in the affinity of mAb 5C3 for TrkAreceptors. Similar data was obtained measuring mAb 5C3 binding sites byFACScan analysis, where a decrease was observed after NGF treatment(Table 2).

TABLE 2 mAb 5C3-induced Trka receptor internalization TREATMENT TEMP. (°C.) % 5C3 STAINING NGF (2 nM)  4° C. 83 ± 2.0 37° C. 75 ± 3.6 5C3 (0.01μg/ml)  4° C. 96 ± 9.0 37° C. 77 ± 5.5

TrkA surface immunostaining was performed on 4-3.6 cells with mAb 5C3after the indicated treatments, and measured by FACScan analysis. Datais presented as % staining±sem, with reference to control vehicletreatment (100%) as per the following formula:

(Treated sample staining−mIgG background staining)×100% (maximumstaining−mIgG background staining)

In the converse experiment, mAb 5C3 inhibited ˜60% of ¹²⁵I[NGF] bindingto E25 cells. In these experiments background binding was assessed byblocking with 5 μM NGF (100% inhibition), and maximal binding wasassessed with binding buffer vehicle only (0% inhibition) or by usingirrelevant binding mAb 87.92.6 (Table 3).

TABLE 3 MAb 5C3 blocks NGF binding to TrkA TREATMENT % NGF BINDING mAb5C3 39.3 ± 7.4 mAb 87.92.6 100 NGF (5 μM)  0

E25 cells expressing TrkA (but not p75 receptors) were incubated with¹²⁵I[NGF] in the presence of the indicated agents. ¹²⁵I[NGF] bindingafter treatment with mAb 87.92.6 was identical to treatment with vehiclebinding buffer. Assays were done 3 times in duplicate. Data is expressedas % binding±standard deviation (sd), where mAb 87.92.6 is maximum and 5μM NGF is background binding as per the formula:$\frac{\left( {{test} - {background}} \right)*100\%}{\left( {{maximum} - {background}} \right)}$

Proliferation/survival Assays

5,000 cells/well in serum-free media (SFM) (GIBCO) supplemented with0.1% BSA were added to 96 well plates (Falcon, Lincoln Park, N.J.)containing serial dilutions of NGF, mAb 5C3, control mAbs, mAb 5C3F_(ab) fragments, control mAb 192 F_(ab) fragments or serum (final 5%FBS, normal growth conditions). Where indicated, F_(abs) were externallycross-linked with goat anti-mouse F_(ab) (GαmF_(ab), Sigma). Wellscontaining all culture conditions but no cells were used as blanks. Theproliferative/survival profile of the cells was quantitated using thetetrazolium salt reagent (MTT, Sigma) 48-72 hours after plating asinitially described by T. Mosmann (Hansen, M. B. et al.(1989) Jour.Immunol. Meth., 119: 203-210). Optical density readings of MTT were donein an EIA Plate Reader Model 2550™ (Bio-Rad) at 600 nm with the blankssubtracted. Assays were repeated at least 5 times in quadruplicates.

Foci Formation Assays

15×10⁴ E25 cells were plated in a 25% serum-containing 0.35% soft agarmixture in the presence of either mIgG control (0.5 μg/ml), mAb 5C3 (0.5μg/ml), or NGF (2 nM). Conditions were replenished every 3 days and fociwere counted after two weeks.

Immunocytochemistry of Human Brain Tissues

Human brain tissue was obtained from six males (age 71.7±4.6) withoutsigns of neurologic or psychiatric disorders. Tissue blocks wereprepared (mean time post-mortem 16.2±3.5 hrs) and stored at −80° C.Twenty μm thick cryostat sections were fixed (4% paraformaldehyde, 0.1 Mphosphate, pH 7.4; 1 hour at 4° C.), and rinsed in PBS for 1 hour at 4°C. Immunocytochemistry was performed using avidin-biotin complex(Vectastain Elite™ kit, Vector Labs) as described (Hsu, S-M. et al.(1981) J. Histochem. Cytochem., 29: 577-580). Primary mAb 5C3 was usedeither as a 1:1000-1:4000 dilution of ascites or a 1:4 dilution of serumfree-media culture supernatant. Where indicated, 0.5% nickel ammoniumsulfate was used to amplify the signal in the DAB revelation step. Somesections were also stained with cresyl violet to facilitate thecytoarchitectural analysis. Negative controls were performed withoutprimary antibody or with normal mouse IgG as primary and in all casesyielded no detectable immunolabeling.

Immunostaining in Normal Human Brain

MAb 5C3 was used to map TrkA protein expression by immunocytochemistryof normal adult human brains. The striatum, the basal forebrain and thebrainstem exhibited the strongest immunostaining, whereas only weakstaining could be detected in the cerebral cortex and hippocampalformation (FIG. 3).

FIGS. 3A and 3B illustrate low power photomicrographs of the nucleusbasalis of Meynert showing large neurons (arrows) immunoreactive withmAb 5C3 (FIG. 3A) but lacking immunoreactivity with normal mouse IgG(FIG. 3B) in a consecutive section. Note in A that the labeled neuronalprocesses can often be followed (small arrows). Scale bar=50 μm.

FIGS. 3C, 3D, and 3E illustrate high power photomicrographs ofTrkA-containing neurons in the nucleus basalis (FIG. 3C), the putamen(FIG. 3D), and the CA4 sub-field of the hippocampus (FIG. 3E). Theperinuclear area displayed particular strong concentration of DABprecipitate (small arrows) often in granules. Labeled proximal processescould also be observed (arrows). n, nucleus. Scale bars=10 μm.

FIG. 3F illustrates in the pontine nuclei many weakly to stronglystaining neurons (arrows) within the fiber network (small arrows) andaround the non-labeled fiber bundles (FIG. 3F). Scale bar=20 μm.

FIG. 3G illustrates in the reticular formation of the brainstem numerousfibers (small arrows) constitute a network where some scattered neurons(arrow) are observed. Scale bar=20 μm.

FIG. 3H illustrates a photomicrograph of TrkA immunoreactivity in thefrontal cerebral cortex showing weak labeling. A few neurons are weaklypositive (arrows) with the staining residing mostly in puncta possiblycorresponding to fibers (small arrows). Scale bar=10 μm.

All sectors of the basal nucleus contained large TrkA-positive neurons(FIGS. 3A and 3C), most of them in groups embedded in a dense network ofoverlapping stained processes (FIG. 3A). The cells had heterogeneousshapes, ranging from complex multipolar to fusiform.

In the basal ganglia TrkA was detected in distinct cellularcompartments. The caudate nucleus, the nucleus accumbens and the putamencontained several immunoreactive cell bodies without apparentdistinction in density, perikaryal staining and shape. FIG. 3D showstypical labeled multipolar neurons that displayed strong granularimmunoreactivity around the nucleus and in proximal processes. Moreover,numerous puncta and varicose fiber fragments were observed in theseareas. The globus pallidus and the claustrum were mostly negative exceptfor varicose fibers. Similarly, the interstitial elements and fiberbundles did not contain reactive fibers whereas the internal capsuledisplayed some labeled punctas and fibers particularly near the putamenand caudate nucleus.

The hippocampal formation showed weak immunostaining located principallyin scattered fibers and puncta in the stratum granulosum of the dentategyrus, as well as in the strati oriens and pyramidal of the Ammon'shorn. In addition, some weakly stained perikarya could be observed inthe stratum pyramidal of the CA2 and CA3 sub-fields of the Ammon's Hornand in the hilus of the dentate gyrus (CA4 sub-field, FIG. 3E). Theperikarya of these neurons was relatively large in size, of ovoid topyramidal shape, and bearing one prominent apical and radial dendriticprocess. The immunoreactivity appeared like in other stained cell typesof the brain as small granular patches of precipitate locatedprincipally near the nuclear envelope and in some cases within thecytoplasm (FIG. 3E).

Within the cerebral cortex, particularly in the frontal area, TrkAimmunoreactivity appeared more discrete. At high magnification,immunoreactive puncta and fiber fragments without a particular patternof distribution are observed in all layers, but the laminae III-VIappeared more stained than superficial ones (FIG. 3H). Occasional,weakly staining, medium-sized perikarya were observed in layer IV (FIG.3H).

In the brainstem TrkA staining is also detected. The pontine nucleicontained numerous immunoreactive medium sized globular perikarya andfibers between the pontocerebellar fibers (FIG. 3F). The reticularformation also displayed strong immunoreactivity for TrkA principallylocated in fiber networks (FIG. 3G). Some large neurons of bipolar ormultipolar shape are also stained. No TrkA immunostaining was observedin the cerebellum.

Functional Agonism of mAb 5C3

Several functional assays of NGF bioactivity were used to test theagonistic potential of mAb 5C3.

Receptor Internalization

4-3.6 cells were treated with TrkA ligands at internalization permissivetemperatures (37° C.) or at non-permissive temperatures (4° C.) (Table2). NGF treatment reduced the % staining of mAb 5C3 to surface TrkA atboth temperatures. Loss of surface 5C3 binding sites suggest directblocking by NGF (FIG. 5). In contrast, mAb 5C3 treatment reduced thenumber of surface 5C3 binding sites only at 37° C. This is likely due toreceptor internalization, which does not occur efficiently at 4° C.Treatment with mIgG or binding buffer control did not reduce the numberof surface 5C3 binding sites at either temperature. Similar data wasobtained with E25 cells.

Receptor Phosphorylation

Anti-phosphotyrosine western blots of E25 or 4-3.6 whole cell detergentextracts revealed that TrkA phosphotyrosinylation (PY) increasedsignificantly over basal levels after short treatment with mAb 5C3 orwith NGF (FIG. 4).

E25 cells were untreated (lane 1) or treated with mAb 5C3 (lane 2), NGF(lane 3), or mIgG (lane 4) for 15 min. at 37° C. Whole cell lysates wereresolved in a 8% SDS-PAGE under reducing conditions and immunoblottedwith anti-phosphotyrosine mAb 4G10. A parallel gel under non-reducingconditions immunoblotted with mAb 5C3 (not shown) controlled for Mr andequal loading of TrkA on all samples.

Densitometric analysis of several blots from E25 and 4-3.6 cells ispresented in Table 4.

TABLE 4 TrkA tyrosine phosphorylation by mAb 5C3 TREATMENT E25 cells4-3.6 cells mAb 5C3 2.7 ± 0.6 3.4 ± 1.5 NGF 6.5 ± 1.3 3.8 ± 0.8

E25 or 4-3.6 cells were untreated or treated with saturatingconcentrations of mAb 5C3 or NGF for 15 min. at 37° C. Whole celllysates, or anti-PY immunoprecipitates were resolved by SDS-PAGE underreducing conditions, western transferred, immunoblotted withanti-phosphotyrosine (αPY) mAb 4G10, and developed using ECL techniques.Optical density (O.D.) readings were taken from X-ray films with filmbackgrounds subtracted (see Materials and Methods). Data is presented asfold increase in PY of TrkA with respect to untreated cells±sd. n=3.

Other proteins evidence increased PY, including ˜95 kDa and ˜60 kDaproteins, and the p85 subunit of PI-3 kinase (˜2.5-fold increase). Wehave estimated that <10% of all p85 material was tyrosine phosphorylatedupon ligation of TrkA.

Increased Cellular Transformation

NGF treatment causes the transformation and an increase inanchorage-independent growth of TrkA-expressing E25 cells. MAb 5C3caused a ˜2-fold increase in the number and the size of foci as comparedwith mIgG treated cells (Table 5).

TABLE 5 MAb 5C3-induced anchorage-independent growth TREAT- AVERAGENUMBER TYPICAL FOLD INCREASE MENT OF FOCI^(a) CELLS/FOCI^(a) IN FOCI^(b)mIgG 416 ± 45  ˜24   1 ± 0.11 mAb 5C3 806 ± 178 >48  1.9 ± 0.22 NGF 676± 51  ˜24  1.6 ± 0.08

E25 cells were cultured in soft agar in the presence of the indicatedagents for 2 weeks. ^(a)Average number and typical size of foci±sd areshown. ^(b)Fold increase in foci was calculated with respect to mIgGtreated cells (no increase). n=2.

No change in the number or size of foci was observed in wild typeNIH-3T3 cells upon mAb 5C3 treatment.

Protection from Cell Death

Agonistic ligands of TrkA protect receptor-expressing cells from deathin serum-free media (SFM). Both NGF and mAb 5C3 and 5C3 fragments orderivatives increased the number of surviving/proliferating E25fibroblastoid cells (FIG. 6) and 4-3.6 cells (FIG. 8).

Cells were cultured in Serum Free Media supplemented with the indicatedconditions for 2-3 days, followed by the MTT assay. Similar data wasobtained with neuronal 4-3.6 cells (FIG. 8). The %proliferation/survival was determined by standardizing serum containingwells to 100% with the use of the following formula:$\frac{\left( {{O.D.\quad {of}}\quad {test}} \right) \times 100\%}{\left( {{O.D.\quad {of}}\quad {serum}} \right)}$

Equivalent protection was also afforded by TrkA ligands to neuronal4-3.6 cells. In most experiments mAtb 5C3 protection is dose-dependent,although high dose antibody inhibition is sometimes seen (e.g. 1 mg/mlof mAb 5C3).

To ascertain whether cell death is apoptotic, DNA was prepared fromserum-free cultured cells which showed a typical apoptotic fragmentationladder. The DNA ladder was not seen in preparations from cells culturedin the presence of mAb 5C3 or NGF.

Controls demonstrated the functional specificity of mAb 5C3. First,neither NGF nor mAb 5C3 protected wild type NIH-3T3 cells. Second, PC12cells were not protected by mAb 5C3 but were protected by NGF. Third,irrelevant mIgG, GαmF_(ab), or mAb 192 did not protect E25 cells (FIG.6), or NIH-3T3 cells, or 4-3.6 cells (FIG. 8).

Functional Agonism of Monomeric 5C3 Fabs

Monovalent agents that bind TrkA behave as competitive antagonists(Clary, D. O. et al. (1994) Mol. Biol. Cell., 5: 549-563; LeSauteur, L.et al.(1995) J. Biol. Chem. , 270:6564-6569) likely because they can notinduce receptor dimerization. Therefore, it would be expected thatmonomeric 5C3 F_(abs) would be monovalent and not be able to mediateagonistic function.

MAb 5C3 F_(abs) afforded protection from apoptotic death to E25 cells(FIG. 6) and 4-3.6 cells in serum-free media. Moreover,anti-phosphotyrosine western blots revealed that cells treated with 5C3F_(abs) had increased TrkA tyrosine phosphorylation similar to increasesobtained with whole mAb 5C3.

Monomeric 5C3 F_(ab) protection was dose-dependent. However, equivalentor better protective effects were achieved when F_(abs) were externallycross-linked with GαmF_(ab) antibodies. Specificity controls includedthose described in the previous section for whole mAb 5C3, plus 192F_(abs) which had no protective activity in E25 cells.

DISCUSSION

The availability of antibodies against p140 TrkA and p75 has allowed thestudy of these NGF receptors. The mAb 5C3 reported herein is specificfor human TrkA, functions in FACScan immunofluorescence analysis,immunoprecipitation, western blot analysis, and immunocytochemistry.Moreover, mAb 5C3 is a structural and functional mimic of NGF.

Aberrant expression of trkA mRNA and NGF responsiveness have beencorrelated with neurodegenerative disorders and neoplastic malignancy.Hence, TrkA-binding agents will be useful clinical tools in diagnosis,prognosis and perhaps treatment of these diseases. Indeed, mAb 5C3binding is a positive prognostic marker for certain human neoplasias.

TABLE 6 TrkA expression in neuroblastoma Neuroblastomas* Number PositiveMixed Negative Group 1 60 38 17  5 Group 2 53 13  5 35 *15 samplesrepeated after chemotherapy, at the time of second surgery orrecurrence: 5C3 staining patterns remained unchanged in 14 tumors; 1negative tumor subsequently positive post chemotherapy in regions ofmaturing elements.

TABLE 7 TrkA expression in other malignant tumors Malignant tumor N = 42TrkA-Pos Central nervous system tumors 6 0 Rhabdomyosarcomas 5 0Primitive neuroectodermal tumors 6 0 Ewing's sarcomas 2 0 Wilm's tumors6 1 Osteosarcomas 4 0 Melanomas 5 0 Breast carcinomas 5 0 Lungcarcinomas 3 0

TABLE 8 TrkA detection by immunocytochemistry, RT-PCR and western blotIMMUNOCYTO. N RT-PCR Pos N West. Pos Group 1 5C3 Pos 15  15  2 2 5C3 Neg4 4 2 1 5C3 Mixed 1 1 10  10  Group 2 5C3 Pos 11  11  11  11  5C3 Neg11  4 9 5 5C3 Mixed 2 2 0 0

MAb 5C3 recognizes a disulfide-stabilized domain of TrkA and anextracellular epitope with these characteristics appears to be the NGFdocking site. Cross-blocking studies indicated that mAb 5C3 and NGF canreciprocally block each other's binding to TrkA, further suggesting thatthe docking site of 5C3 may be similar to NGF. In addition, sequencecomparison of both ligands revealed interesting homology between CDRs ofmAb 5C3 and the variable turn regions of NGF. Since most CDRs areβ-turns (Sibanda, B. L. et al. (1989) J. Mol. Biol., 206: 759-777) andcoincidentally the NGF structures that bind TrkA may also be β-turns(LeSauteur, L. et al.(1995) J. Biol. Chem., 270:6564-6569) wehypothesized that both mAb 5C3 and NGF bind to the same site on humanTrkA, and cross-blocking is likely to be the result of directcompetition rather than steric hindrance.

Interestingly, mAb 5C3 was more efficient at blocking NGF binding thanvice versa. Only ˜25% of the mAb 5C3 binding sites on E25 cells wereblocked by saturating doses of NGF. This data suggest that not all TrkAreceptors in these cell lines bind NGF. It is unlikely that affinityconsiderations can account for these observations, as both ligands haveroughly comparable K_(d) for TrkA (mAb 5C3 K_(d) ˜2 nM versus NGF K_(d)0.7 nM (Jing, S. et al. (1992) Neuron, 9: 1067-1079)) and the affinityof mAb 5C3 was unchanged in the presence of NGF.

Three non-exclusive possibilities can account for these observations:(i) TrkA receptors exist at equilibrium where ˜25% are in an NGF bindingconformation (e.g. dimers) and the rest are in a non-NGF bindingconformation; (ii) specific post-translational modifications of TrkAreceptors allow for NGF binding (iii) expression of other membraneproteins (e.g. p75 or unknown proteins) induce or favor the NGF bindingconformation of TrkA. These hypotheses can be addressed by biochemicalanalysis after differential affinity purification of TrkA with mAb 5C3versus NGF and by further binding studies in neuronal andfibroblastoid-cells expressing different receptors.

Absence of mAb 5C3 binding to rat TrkA is intriguing. Binding by mAb 5C3to rat TrkA was expected because of the homology between mAb 5C3 CDRsand the variable loops of NGF, particularly since NGF from one speciesdoes bind to TrkAs from other species. MAb 5C3 is a binding andstructural mimic of NGF, with enhanced human receptor specificity.Remodeling and mutating of the CDRs of mAb 5C3 will yield a pan-TrkAbinding mAb. Further, analysis of the epitope of mAb 5C3 on TrkArevealed differences in the docking site of human and rat TrkAs. Thisinformation will be useful in screening receptor-binding analogs.

To test functional mimicry by mAb 5C3, NGF bioassays were performedusing trkA transfected fibroblast and neuronal cells. Functional mimicryby mAb 5C3 included TrkA internalization, TrkA tyrosine phosphorylation,PI-3 kinase phosphorylation, increased anchorage-independent growth andproliferation/survival of cells in serum-free media. By these criteriamAb 5C3 is agonistic.

Increased TrkA receptor turnover or internalization is induced by NGFbinding. MAb 5C3 increased the internalization of TrkA, as measured byloss of cell surface receptors. These results are consistent with datawhich showed that E25 cells internalize ¹²⁵I[NGF] within seconds uponshifting from 40° C. to 37° C. (Jing, S. et al. (1992) Neuron, 9:1067-1079) and that this process does not require p75 receptors. Thus,artificial ligands of TrkA can induce receptor internalization and couldbe useful in delivering toxic agents to the cytoplasma ofTrkA-expressing tumors.

NGF ligation of TrkA causes receptor activation and autophosphorylation.MAb 5C3 induced TrkA tyrosine phosphorylation to a similar degree.Agonism in the absence of NGF suggests that TrkA dimerization and/orinternalization are the required signaling event, rather than theformation of NGF-TrkA complexes. However, we can not rule out that mAb5C3-TrkA is the functional signal transducing complex.

Ligand-induced tyrosine phosphorylation of the intracellular domain ofTrkA allows for the recruitment of substrates and the activation ofcytosolic proteins and nuclear oncoproteins. MAb 5C3 induces thetyrosine phosphorylation of proteins of M_(r) 60, 85 and 95 kDa. The 85kDa protein was identified as PI-3 kinase, whose activation correlateswith the actions of growth factors and oncogenes.

NGF stimulates neuronal survival and differentiation, and theproliferation of non-neuronal cells. NGF-activated TrkA inducestransformation and morphological changes in fibroblast cells. MAb 5C3caused similar increases in anchorage-independent growth and fociformation in soft agar. Thus, mAb 5C3 can positively modulate the growthof TrkA-expressing cells. Interestingly, the size of the mAb 5C3-inducedfoci were larger on average than NGF-induced foci.

TrkA-expressing neuronal 4-3.6 cells or fibroblastoid E25 cells undergoapoptotic death in serum free media but can be rescued by NGF or mAb5C3. Synergy between the two ligands occurred when combined atsuboptimal doses, as would be expected if mAb 5C3 bound and activatedunoccupied TrkA receptors. Furthermore, morphological changes andincreased attachment to plastic were observed in both the NGF and 5C3treated cells.

Monomeric 5C3 F_(abs) protected E25 and 4-3.6 cells from apoptoticdeath. When F_(abs) were externally cross-linked using anti-F_(ab)antibodies, a heightened response occurred. Since growth factor receptoractivation requires bivalent binding, the monomeric 5C3 F_(abs) musthave retained the ability to induce TrkA oligomerization. This could beexplained in the following 3 ways: (i) F_(abs) are relatively largemolecules capable of aggregation; (ii) 5C3 F_(ab) binding, could causeconformational changes in TrkA which induce receptor-receptorinteractions; and (iii) monomeric 5C3 F_(abs) bind to two receptormolecules in a bivalent manner. The last possibility could occur by twoCDRs binding to two different TrkAs. Homology of mAb 5C3 CDRs to NGFturn regions, and experiments using small recombinant antibody analogssupport the last explanation.

MAb 5C3 is the first report of an agonistic anti-neurotrophin receptormAb and will be useful in studies of TrkA biology and for drugdevelopment. Antineoplastic effects with mAb 5C3 may be achieved eitherthrough terminal differentiation, antibody-dependent cell cytotoxicity,or by the delivery of toxins or radionuclides. Furthermore, thestructure of this mAb may be useful in designing peptidic andnon-peptidic TrkA binding agents (Saragovi, H. U. et al. (1991) Science,253: 792-795). Small non-peptidic agonists of TrkA should be usefulpharmacological agents for the treatment of neurodegenerative diseases.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

EXAMPLE I Preliminary Study of the Effect of mAb 5C3 in Tumor Growth

Nude mice were injected subcutaneously (right abdominal side) with 2×10⁶human TrkA expressing tumor cells. Two days post-injections tumors inall mice had begun to form. Mice were randomized prior to treatment. Atotal of four intraperitoneal injections of 100 micrograms each on theleft side were then administered over a 12 day period with control mouseIgG or mAb 5C3 (FIG. 10).

The mAb 5C3 dramatically reduced the primary tumor weight with noobservable metastatic invasion. A small fibrotic mass was localized atthe site of injection in mAb 5C3 treated mice. In contrast, IgG treatedmice had large, vascularized tumor masses, which metastasized to theliver, peritoneum gut and spleen. All animals had similar body weights(˜30 grams).

TABLE 9 PRIMARY METASTASIS TUMOR WEIGHT WEIGHT TREATMENT (mg) (mg) 5C3mAb 50 ± 20 NONE (fibrotic) mouse IgG 800 ± 250 350 ± 20 

EXAMPLE II The Use of Mab 5C3 and Its Derivatives for the Diagnosis,Prognosis and Localization of Tumors

The in vivo targeting efficacy of agents that bind the NGF receptor p140TrkA was evaluated. Nuclear imaging studies were done after theinjection of ^(99m)Tc-labeled compounds in nude mice bearing tumors.Kinetics of tumor targeting, blood clearance, and bioavailability werestudied. Tumors that do not express TrkA were not targeted,demonstrating the specificity in vivo. This biodistribution studydemonstrates that receptor-specific molecule analogs may be useful andmay be effective agents for the detection, diagnosis, and possibletreatment of neoplasias involving overexpressed oncogenic receptors suchas TrkA (FIG. 7).

TABLE 10 Biodistribution of ^(99m)Tc-[TrkA ligands] in mice.^(99m)Tc-[5C3] Ligand % id/g T/nT 1 tumor 1.25 1 2 blood 0.1 13 3 muscle0.06 20 4 heart 0.10 13 5 lung 0.17 7.30 6 liver 0.61 2.10 7 spleen 0.139.42 8 kidney 1.48 0.9 9 large bowel 2.2 0.7

EXAMPLE III 5C3 Protein Sequence

Kappa light chain

DILQTQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPLTFGAGTKLEI  (SEQID NO:1)

Heavy chain (IgG2a)

VQLQESGTVLARPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGAIYPGDSDTSYNQKFKGEAKLTAVTSTSTAYMELSSLTNEDSAVYYCTLYGNYESYYAMDYWGQGILSHRLL  (SEQID:2)

Complementarity Determining regions (according to Kabat, Sequences ofProteins of Immunological Interest, 4th ed. Bethesda, Md, U.S. Dept. ofHealth and Human Services. Public Health, NIH 1987)

Kappa variable light chain

VL CDR1:RASSSVSYMH  (SEQ ID NO:3)

VL CDR2:ATSNLAS  (SEQ ID,NO:4)

VL CDR3:QQWSSNPLT  (SEQ ID NO:5)

Heavy chain variable (IgG2a)

VH CDR1:SYWMH  (SEQ ID NO:6)

VH CDR2:AIYPGDSDTSYNQKFKG  (SEQ ID NO:7)

VH CDR3:YGNYESYYAMDY  (SEQ ID NO:8)

Antibodies have 6 complementarity determining regions (CDRs) thatcombine in specific ways to create direct contacts with their antigen.Molecular cloning, recombination, mutagenesis and modeling studies ofmAb 5C3 variable region indicated that 3 or less of its CDRs arerelevant for binding TrkA. These were named CDR1, CDR2, and CDR3. RegionCDR1 is connected to CDR2 by a 15 amino acid linker; CDR2 is connectedto CDR3 by a 30 amino acid linker. Their secondary structures have beenanalyzed.

The variable domains of mAb 5C3 heavy (VH) and light (LH) chains werecloned and sequenced. The predicted variable domain amino acid sequencesare shown above.

The hypervariable complementarity determining regions (CDRs) wereresolved according to Kabat (Sequences of Proteins of ImmunologicalInterest, 4th ed. Bethesda, Md., U.S. Dept. of Health and HumanServices. Public Health, NIH 1987).

An example of the model of the structure of the CDRs (Insight II™,Biosym, Calif.) within the backbone of the variable domains is shown inFIG. 11.

EXAMPLE IV MAb 5C3 and Derivatives: Structural and Functional Mimics ofNGF

Several criteria indicate that mAb 5C3 is a TrkA agonist. Previousfunctional studies in vitro demonstrated that mAb 5C3 can: (i) inducetyrosine phosphorylation of TrkA, and PI3-kinase; (ii) increasetransformation of TrkA-expressing fibroblasts. Shown above are alsoexperiments where the 5C3 and its derivatives protect cells fromapoptotic death in serum-free media to the same degree as NGF does.Monovalent Fabs of 5C3 obtained after papain digestion are alsoagonistic, especially when externally cross-linked by anti-Fabs. Asmaller fragment of mAb 5C3 called CDR(R) also protects cells fromapoptosis (FIG. 8).

Assays with recombinant CDRs and CDR-like synthetic polypeptidesdemonstrated they had agonistic bioactivies similar to intact mAb 5C3.Consequently a 6 kDa recombinant molecule “CDR(R)” (for recombinant CDR)was developed as a replacement for mAb 5C3; it is approximately fivetimes smaller than the 25 kDa NGF molecule and is still agonistic.CDR(R) is composed of 3 selected CDRs (out of 6 possible ones) linked bylong spacer regions. Preliminary studies have suggested that actuallyonly 2 of the 3 CDRs are relevant for binding to TrkA. Further, it isexpected that even smaller fragments can be designed, e.g. upon removalof the linker regions.

4-3.6 cells transfected with human TrkA were cultured inserum-free-media (SFM) to induce apoptosis (FIG. 8). The indicatedagents were added to the cells and cell viability was measured after48-72 h by the MTT assay. Proliferation was standardized to normalgrowth conditions (5% serum). NGF, mAb 5C3, 5C3 Fabs, and CDR(R)(recombinant 5C3 CDR analog) protect from apoptosis, but mouse IgG(control) does not.

EXAMPLE V Use of Mab 5C3

The mAb 5C3 of the present invention and its derivatives inducedifferentiation/neuritogenesis of human TrkA-expressing cells. PC12cells (expressing rat TrkA) and PC12 cells transfected with andexpressing human trkA cDNA were cultured with various TrkA bindingagents or controls (FIG. 9) as indicated NGF caused the differentiationof wild type PC12 and PC12 transfectants, whereas SC3 and CDR(R) onlyinduced differentiation of PC12 transfectants. Ligand doses are as shownin FIG. 8.

Further modifications of CDR(R) resulted in agents that support cellsurvival but not neuritogenesis.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

9 105 amino acids amino acid linear protein 1 Asp Ile Leu Gln Thr GlnSer Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr MetThr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln GlnLys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Thr Ser Asn LeuAla Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr SerTyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala ThrTyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala GlyThr Lys Leu Glu Ile 100 105 120 amino acids amino acid linear protein 2Val Gln Leu Gln Glu Ser Gly Thr Val Leu Ala Arg Pro Gly Ala Ser 1 5 1015 Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp 20 2530 Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 35 4045 Ala Ile Tyr Pro Gly Asp Ser Asp Thr Ser Tyr Asn Gln Lys Phe Lys 50 5560 Gly Glu Ala Lys Leu Thr Ala Val Thr Ser Thr Ser Thr Ala Tyr Met 65 7075 80 Glu Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Thr 8590 95 Leu Tyr Gly Asn Tyr Glu Ser Tyr Tyr Ala Met Asp Tyr Trp Gly Gln100 105 110 Gly Ile Leu Ser His Arg Leu Leu 115 120 10 amino acids aminoacid linear protein 3 Arg Ala Ser Ser Ser Val Ser Tyr Met His 1 5 10 7amino acids amino acid linear protein 4 Ala Thr Ser Asn Leu Ala Ser 1 59 amino acids amino acid linear protein 5 Gln Gln Trp Ser Ser Asn ProLeu Thr 1 5 5 amino acids amino acid linear protein 6 Ser Tyr Trp MetHis 1 5 17 amino acids amino acid linear protein 7 Ala Ile Tyr Pro GlyAsp Ser Asp Thr Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly 12 amino acidsamino acid linear protein 8 Tyr Gly Asn Tyr Glu Ser Tyr Tyr Ala Met AspTyr 1 5 10 131 amino acids amino acid linear protein 9 Gln Val Asn ValSer Phe Pro Ala Ser Val Gln Leu His Thr Ala Val 1 5 10 15 Glu Met HisHis Trp Ser Ile Pro Phe Ser Val Asp Gly Gln Pro Ala 20 25 30 Pro Ser LeuArg Trp Leu Phe Asn Gly Ser Val Leu Asn Glu Thr Ser 35 40 45 Phe Ile PheThr Glu Phe Leu Glu Pro Ala Ala Asn Glu Thr Val Arg 50 55 60 His Gly CysLeu Arg Leu Asn Gln Pro Thr His Val Asn Asn Gly Asn 65 70 75 80 Tyr ThrLeu Leu Ala Ala Asn Pro Phe Gly Gln Ala Ser Ala Ser Ile 85 90 95 Met AlaAla Phe Met Asp Asn Pro Phe Glu Phe Asn Pro Glu Asp Pro 100 105 110 IlePro Asp Thr Asn Ser Thr Ser Gly Asp Pro Val Glu Lys Lys Asp 115 120 125Glu Thr Pro 130

We claim:
 1. A monoclonal antibody comprising SEQ ID NOS: 3, 4, 5, 6, 7,and
 8. 2. A method of in vitro screening for an agent which is capableof activating Trka receptor, which comprises combining an antibody ofclaim 1 comprising SEQ ID NOS: 3, 4, 5, 6, 7, and 8 with TrkA receptorand a candidate agent and detecting reduced binding of said antibody toTrk A receptor, wherein reduced binding is indicative of binding to TrkAreceptor by said candidate agent, indicating that said agent is capableof binding to TrkA receptor and thereby activating TrkA receptor.
 3. Amonoclonal antibody of claim 1, said antibody or an antigen bindingfragment thereof capable of activating TrkA receptor, said antibody orantigen binding fragment thereof being raised against human TrkAreceptor and binding specifically to TrkA receptor under physiologicalconditions, wherein said binding to said receptor activates the TrkAreceptor.
 4. A monoclonal antibody of claim 1, said antibody or antigenbinding fragment thereof being raised against human TrkA receptor andspecifically binding to TrkA receptor or to the IgG2 domain of TrkAreceptor under physiological conditions, and wherein said binding tosaid domain activates TrkA receptor.
 5. A monoclonal antibody of claim 1or an antigen binding fragment thereof raised against human TrkAreceptor and binding specifically to TrKA receptor under physiologicalconditions, and wherein said binding to said receptor activates TrkAreceptor.